CN103289905B - Low-rank coal degrading bacteria and application thereof in brown coal liquefaction - Google Patents
Low-rank coal degrading bacteria and application thereof in brown coal liquefaction Download PDFInfo
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Abstract
The invention discloses application of low-rank coal degrading bacteria Davidiellatassiana CGMCC No.7440 in brown coal liquefaction. The optimum growth conditions of a bacterial strain are as follows: the temperature is 25 DEG C, and the culture medium is CDA; the cultivation condition is as follows: the pH is 6.5, and the cultivation time is 24-48 hours. Cultivation is carried out for 48 hours at 25 DEG C on the CDA solid culture medium; the bacterial colony is characterized by being a yellow-green colony which is about 1cm in diameter and forms a round-like shape; the surface forms a napo shape; moundy raised drapes are formed on the surface; the edge forms a cilium shape; and the cilium is loose in character and easy to be picked. The taxonomic status is primarily determined by carrying out morphological characteristics and ITS (internal transcribed spacer) sequence determination analysis on the obtained bacterial strain; a new choice is provided for the low-rank coal degrading bacteria; the degradation has an outstanding technical effect on solving of low-rank coal liquefaction and has an important role.
Description
Technical field
The present invention relates to microbiological deterioration low-rank coal technical field, concrete, the present invention relates to a kind of technical field of utilizing degraded low-rank coal bacterium and the application in brown coal liquefaction thereof.
Background technology
In state-owned abundant coal resources, explored coal reserves exceedes 8.1 × 10
11t accounts for more than 70% in China's Energy Mix, and 75% fuel used to generate electricity, 75% industrial fuel, 80% resident living fuel and 60% industrial chemicals, be all from coal.Xinjiang is as the important coal place of production of China, and coal resources are very abundant, and 1,008 hundred million tons of proven reserve account for the whole nation 10%, and 2.19 trillion tons of prognostic reserves, account for the more than 40% of national total reserves, rank first in the country.
Low-rank coal is the coal in low metamorphic episode, according to Chinese coal seam coal classification GB/T17607 classification, the definition of low-rank coal is the coal of Qgr.maf<24 MJ/kg, coal class is divided into lower-grade metamorphic bituminous (comprising long-flame coal, non caking coal, weakly caking coal) and brown coal.The low-rank coal such as weathered coal, brown coal resource, in China, explored brown coal retained reserve reaches 1,303 hundred million tons, account for national coal reserves 13% a little less than.Brown coal are that a kind of metamorphic grade is low, easy-weathering spontaneous combustion, thermal value are low, water content, oxygen level and the higher young coal of volatile matter, and aromatic series carbon content is lower, and contains a small amount of element such as nitrogen and sulphur.Direct combustion thermal efficiency of coal resource is low at a low price for these, and industrial application value is low, and long-term air storage, not only causes the waste of the energy, and easily cause environmental pollution.Therefore, how reasonable development and make full use of brown coal and low-rank coal resource will be a problem that is worth further investigation.
Coal is as resource, and tool has been widely used, and both can be used as the energy, can extract again useful Chemicals, and more than 60% industrial chemicals, just from coal, plays an important role to expanding economy.As the energy, the necessary clean and effective utilization of coal, adopts the means such as high temperature, high pressure that coal is changed into other class A fuel A such as liquid, gas and replaces oily substance, is exactly the one that its Efficient Conversion utilizes.And from coal, extract Chemicals, what conventionally adopt is that the condition of the certain temperature of additional certain pressure physics, chemistry gets off to carry out.Be oily substance and the process of extracting chemicals from coal conversion, cost is higher, condition is harsh.And adopt microbiological transformation technology to process coal, make it to change into another kind of product, or as fuel, or therefrom extract chemicals, or as other class material, have advantages of that technique is simple, less energy-consumption, many Technology developments such as pollution-free hardly match.Therefore, microbiological transformation technology is processed coal has become the focus of domestic and international research.Utilize white-rot fungi, actinomycetes etc. carry out low-rank coal liquefaction report, study more, but utilize
davidiella tassianathe research that bacterial classification carries out low-rank coal liquefaction have not been reported.
Summary of the invention
In view of many, the traditional utility values of the current brown coal output of prior art, low, long-term stacking causes the state of the art of environmental pollution, and the present invention utilizes the advantage of microbiological transformation technology, and the problem that is intended to solve is that the microorganism providing is provided
davidiella tassianadegraded brown coal, and obtain good effect.
The present invention adopts main technical scheme:
By to the screening of carrying out low-rank coal degradation bacteria in the coal sample sample in region, colliery, Fukang, Xinjiang city, obtain have stronger liquefying activity bacterial strain, through further screening, domestication breeding, obtain a strain and be numbered the bacterial strain of RM-1.By obtained bacterial strain being carried out to morphological specificity and ITS Sequence analysis, tentatively determine its classification position; For low-rank coal degradation bacteria provides new selection, its Degradation has outstanding technique effect for the solution of low-rank coal liquefaction.
A kind of low-rank coal degradation bacteria that the present invention specifically provides, by separating in the coal sample sample in region, colliery, Fukang, Xinjiang city, screen and cultivating, obtain a collection of microorganism strains as degradable low-rank coal, therefrom filter out the bacterial strain that a strain is numbered RM-1, degraded Sha Er lake brown coal have to be stablized and significant effect, through microbiology classification and identification, belong to
davidiella tassiana.
Concrete, a kind of low-rank coal degradation bacteria provided by the invention
davidiella tassianacGMCC No.7440, is numbered RM-1.This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date is on April 9th, 2013, and preserving number is CGMCC No.7440.Be accredited as through microbiology
davidiella tassiana.This bacterial strain optimum growing condition is: 25 ° of C of temperature, substratum is CDA substratum, culture condition: pH:6.5, incubation time: 48-72h.On CDA solid medium, cultivate 48h for 25 ℃, colony characteristics is the yellow-green colour bacterium colony of diameter 1cm left and right, is sub-circular, and surface is short down shape, has mound shape protuberance fold, and edge is cilium shape, and quality is loose, easily provokes; Carry out morphology mensuration according to " fungi identification handbook " to being numbered RM-1 bacterial strain, Physiology and biochemistry detects determines that being numbered RM-1 bacterial strain is
davidiella tassianain member.Compare by BLAST homology, the ITS sequence of bacterial strain RM-1 is carried out after BLAST analysis in ncbi database, constructing system evolutionary tree, bacterial strain RM-1 with
davidiella tassianain minimum branch, it is its allied species; Bacterial strain ITS sequence is committed to
geneBankmiddle acquisition gene accession number is: KF002787.Thereby will be numbered RM-1 bacterial strain and be defined as
davidiella tassiana.
Meanwhile, the invention provides bacterial classification low-rank coal degradation bacteria
davidiella tassianathe preservation culture condition of CGMCC No.7440, CDA solid medium and culture condition: sucrose 3.0%, yeast extract 0.5%, K
2hPO
40.1%, MgSO
47H
2o 0.05%, KCl 0.05%, NaNO
30.3%, FeSO
47H
2o0.001%, agar 2%.Culture condition: 25 ° of C of temperature, pH:6.5, incubation time: 24-48h.In its CDA medium slant, under 25 ° of C conditions, cultivate 24-48 hour, the aseptic skimmed milk of rear employing is protective material, cryopreservation after vacuum lyophilization; Every day use is kept on the inclined-plane of CDA solid medium, and 4 ° of C Refrigerator stores are for subsequent use.
Further, the invention provides low-rank coal degradation bacteria
davidiella tassianacGMCC No.7440 growth conditions, best incubation time is 24h, and the most suitable growth pH is 6.5, and the large volume production spore time is 24h; Determine that optimum carbon source is sucrose, optimum nitrogen source is yeast extract.
The present invention provides low-rank coal degradation bacteria simultaneously
davidiella tassianathe application of CGMCC No.7440 in brown coal liquefaction.By add 150ml CZAPECK DOX-medium substratum in 500ml triangular flask, every bottle adds 5g pre-treatment coal sample.Pretreatment process is for to be crushed to 100 orders by coal sample particle diameter, and under 121 ℃ of conditions sterilizing 30min.Meanwhile, for coal sample is more easily liquefied, coal sample is soaked in the salpeter solution of 6mol/L carries out 48h dipping pretreatment.Finally, bacterial strain RM-1 inoculation is entered in triangular flask, through fermentation in 21 days, measure every strain bacterial strain transformation efficiency by acid precipitation method.And by application Sha Er lake brown coal, large South Lake, Hami long-flame coal etc. has same effect, degraded Sha Er lake brown coal have to be stablized and significant effect, cultivate 72 hours its brown coal of can significantly degrading and produce black drop, verify that through many experiments its degradation effect is comparatively stable, thereby can prove bacterial classification provided by the invention
davidiella tassianathe application of CGMCC No.7440 in brown coal liquefaction obtains significantly and significantly technique effect.
By implementing the concrete summary of the invention of the present invention, can reach following beneficial effect.
(1) the invention provides and separate the bacterial classification obtaining
davidiella tassianacGMCC No. 7440 is a kind of low-rank coal degradation bacteria, by application Sha Er lake brown coal, large South Lake, Hami long-flame coal etc. has same effect, degraded Sha Er lake brown coal have to be stablized and significant effect, cultivate 72 hours its brown coal of can significantly degrading and produce black drop, verify that through many experiments its degradation effect is comparatively stable.
(2) utilize provided by the invention
davidiella tassianacGMCC No. 7440 liquefaction of brown coal effectively can being degraded, and liquefied product is applied in the middle of industrial and agricultural production, the secondary refuse of contaminate environment can not produced at fermenting process.
Accompanying drawing explanation
Fig. 1 is shown as low-rank coal degradation bacteria
davidiella tassianathe colonial morphology figure mono-of CGMCC No. 7440.
Fig. 2 is shown as low-rank coal degradation bacteria
davidiella tassianathe colonial morphology figure bis-of CGMCC No. 7440.
Fig. 3 is shown as low-rank coal degradation bacteria
davidiella tassianathe ITS phylogeny tree graph of CGMCC No.7440.
Fig. 4 is shown as low-rank coal degradation bacteria
davidiella tassianacGMCC No. 7440 state graph before brown coal of degrading.
Fig. 5 is shown as low-rank coal degradation bacteria
davidiella tassianathe degraded brown coal result figure of CGMCC No. 7440.
Embodiment
, for embodiment, the present invention is described below, still, the present invention is not limited to following embodiment.
The coal sample that the present invention adopts: Sha Er lake brown coal; Substratum and other material: substratum is selected: CDA solid, CZAPECK DOX-medium liquid nutrient medium.
Key instrument and reagent: MSSPX-250 type biochemical cultivation case, MLS-3020 high-pressure steam sterilizing pan, the single two-sided clean work station of SW-CJ-1F Type B, E360K whizzer, constant-temperature table HWY-100.PCR instrument Eppendorf No:5345, electrophoresis apparatus Bio-Rad Mode 200/2.0, gel imaging instrument United-Bio, GK-330C plus, PCR premixed liquid (
taKaRa Biotechnology), all the other reagent are analytical pure.
All bacterial classifications and the raw and auxiliary material in the present invention, selected, and the spawn culture condition of selecting and method be all well known selecting, the % relating in the present invention is weight percentage, unless otherwise indicated except.
embodiment mono-: separation, screening, classification and the evaluation of microorganism RM-1 bacterial strain
(1) Isolation and screening of bacterial classification
By separating in the coal sample sample in region, colliery, Fukang, Xinjiang city, screen and cultivating, obtain a collection of microorganism strains as degradable Xinjiang low-rank coal, by further screening, the concrete steps of screening are: get all product of 1g pit coal, add sterilized water, be diluted to respectively 10
0, 10
-1and 10
-2three gradient concentrations, evenly coat on CDA solid medium 25 ℃, leave standstill and cultivate, after bacterium colony is fully grown, select the bacterium colony renewed vaccination of different shape and carry out purifying to CDA solid medium, in 5 generations of purifying so repeatedly, through plate screening, be divided into from obtaining 7 strain brown coal degradation bacteria strains; Through further sieving again, obtain degradation bacteria strains RM-1.Therefrom filter out the bacterial strain that a strain is numbered RM-1, by inoculation to CDA solid medium, be statically placed in 25 ℃ of incubators and cultivate 24-48h, after bacterial strain covers with whole substratum, the coal dust that evenly adds one deck nitric acid treatment to cross, visible RM-1 bacterial strain can be by the degraded of Xinjiang low-rank coal, by application Sha Er lake brown coal, large South Lake, Hami long-flame coal etc. has same effect, produces black drop at RM-1 mycelium surface.Degraded Sha Er lake brown coal have to be stablized and significant effect, cultivates 72 hours its brown coal of can significantly degrading and produces black drop, verifies that through many experiments its degradation effect is comparatively stable.Through microbiology classification and identification, belong to
davidiella tassianaclass.
Concrete, a kind of low-rank coal degradation bacteria provided by the invention
davidiella tassiana, be numbered RM-1.This bacterial strain was preserved in the international depositary institution of budapest treaty microorganism before the applying date: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date is on April 9th, 2013, and preserving number is CGMCC No. 7440.Be accredited as through microbiology
davidiella tassiana.This bacterial strain optimum growing condition is: 25 ° of C of temperature, substratum is CDA, culture condition: pH:6.5, incubation time: 24-8h.On CDA solid medium, cultivate 48h for 25 ℃, colony characteristics is the yellow-green colour bacterium colony of diameter 1cm left and right, is sub-circular, and surface is short down shape, has mound shape protuberance fold, and edge is cilium shape, and quality is loose, easily provokes, referring to accompanying drawing 1; According to " fungi identification handbook ", carry out morphology mensuration to being numbered RM-1 bacterial strain, Physiology and biochemistry detects determines that being numbered RM-1 bacterial strain is
davidiella tassianain member.Compare by BLAST homology, the ITS sequence of bacterial strain RM-1 is carried out after BLAST analysis in ncbi database, constructing system evolutionary tree, shown in accompanying drawing 2, bacterial strain RM-1 with
davidiella tassianain minimum branch, it is its allied species; Bacterial strain ITS sequence is committed to
geneBankmiddle acquisition gene accession number is: KF002787.Thereby will be numbered RM-1 bacterial strain and be defined as
davidiella tassiana.
(2) PCR amplification Antagonistic Fungi ITS sequence and order-checking thereof
The a small amount of single bacterium colony of picking, puts into the EP pipe that fills 25 μ L sterilized waters, and 100 ° of C boil 8-10 min, rear mixture of ice and water 5 min that put into rapidly.Centrifugal 10000 r/min, 5 min, 4 ° of C preserve, and the used time is got supernatant.Pcr amplification ITS sequence.ITS sequential analysis, take the total DNA of cell that extracts as template, utilizes primer:
Upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ';
Downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '.
Pcr amplification reaction system is 50 μ L, contains 24 μ L premix Taq, primer 11 μ L, primer 21 μ L, template 2 μ L, sterilized water 22 μ L.Amplification condition: 94 ° of C 4 min, 94 ° of C 55 s, 53 ° of C 30 s, 72 ° of C 90 s, 30 circulations; 72 ° of C 7 min.Amplified production (approximately 500 bp) is through 1% agarose gel electrophoresis isolation identification, and PCR product directly carries out two-way order-checking.
Pcr amplification product is checked order, after order-checking, confirm that this fragment experiment length is 486bp.
(3)iTS
sequence alignment and Phylogenetic Analysis
Nucleotide sequence in ITS sequence and the GenBank database that order-checking is obtained carries out BLAST analysis, therefrom obtains close ITS sequence, with Clustal X software and MEGA 4.1Neighbor-joining method constructing system evolutionary tree, referring to accompanying drawing 2.Bacterial strain ITS sequence is submitted to and is utilized the BankIt software in GeneBank to submit to online, obtains gene accession number KF002787.
In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of RM-1, determine that it is
davidiella tassianacGMCC No. 7440.
The ITS sequence of RM-1 is carried out to BLAST analysis, result demonstration, the similarity of RM-1 and bacterial strain Davidiella tassiana is the highest, has reached 100%.
The ITS sequence of RM-1 is carried out after BLAST analysis in ncbi database to constructing system evolutionary tree.Shown in accompanying drawing 3, between bacterial strain RM-1 and Davidiella tassiana, evolutionary distance is the shortest, is
davidiella tassianaallied species.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of RM-1, determine that it is
davidiella tassiana.
Comprehensively, according to ITS the sequencing results, combining form and physio-biochemical characteristics, be accredited as bacterial strain RM-1
davidiella tassiana.
embodiment bis-:low-rank coal degradation bacteria
davidiella tassiana the somatomedin of CGMCC No. 7440
Referring to following mode, but should be according to provided by the invention
davidiella tassianacGMCC No.7440 characteristic is determined the somatomedin that it is concrete.
Bacterial strain RM-1 is received to CDA solid medium to be cultivated.The results are shown in Table 1.
Drawn the most applicable somatomedin of bacterial strain RM-1 by table 1.
table 1: temperature, pH, salt, the impact of microbiotic on bacterial strain RM-1 growth
| Temperature (℃) | 4 | 10 | 15 | 20 | 25 | 30 |
| Growing state | + | + | + | + | +++ | ++ |
| Temperature (℃) | 35 | 40 | 45 | 50 | 55 | 60 |
| Growing state | ++ | + | — | — | — | — |
| |
1 | 2 | 3 | 4 | 5 | 6 |
| Growing state | — | — | — | — | + | ++ |
| pH | 7 | 8 | 9 | 10 | ||
| Growing state | + | + | + | — | ||
| NaCl concentration | 0.5％ | 1％ | 2％ | 3％ | 4％ | 5％ |
| Growing state | ++ | ++ | ++ | ++ | + | + |
| NaCl concentration | 6％ | 7％ | 8％ | 9％ | 10％ | |
| Growing state | + | — | — | — | — |
Draw the most applicable somatomedin of bacterial strain RM-1 by table 1.
CDA solid medium and culture condition: sucrose 3.0%, yeast extract 0.5%, K
2hPO
40.1%, MgSO
47H
2o0.05%, KCl0.05%, NaNO
30.3%, FeSO
47H
2o0.001%, agar 2%.Culture condition: 25 ° of C of temperature, pH:6.5, incubation time: 24-48h.
embodiment tri-:low-rank coal degradation bacteria
davidiella tassianaCGMCCno. 7440 brown coal degradation experiment
In order further to detect the liquefying power to Xinjiang low-rank coal in RM-1 liquid medium within, Xinjiang Sha Erhu brown coal are added in liquid nutrient medium.Coal sample particle diameter is crushed to 100 orders, and under 121 ℃ of conditions sterilizing 30min.For coal sample is more easily liquefied, coal sample is soaked in the salpeter solution of 6mol/L carries out 48h pre-treatment.
In 500ml triangular flask, add 150ml CZAPECK DOX-medium substratum, every bottle adds 5g coal sample.Bacterial strain RM-1 inoculation is entered in triangular flask, through fermentation in 21 days, measure every strain bacterial strain transformation efficiency by acid precipitation method.
Acid precipitation method: first use whizzer by the mycelium after fermentation ends and cinder and separation of fermentative broth, re-use qualitative filter paper by filtering fermentation liquor.In the fermented liquid obtaining, add acid, make fermented liquid pH value stabilization below 2, obtain Acid precipitation product.Acid precipitation product is put into 80 ℃ of oven drying 18-20h, be precipitated the dry weight of product, experimental result, in table 2, is calculated transformation efficiency:
Transformation efficiency=(precipitation quality/add coal sample quality) × 100%
Table 2: transformation efficiency test-results
| Project | Substratum | Substratum+bacterium | Substratum+coal | Substratum+coal+bacterium |
| Transformation efficiency (%) | 0 | 0 | 1.5 | 58.34 |
embodiment tetra-:low-rank coal degradation bacteria
davidiella tassianaCGMCCno.7440 takes off cell extract brown coal degradation experiment
For in proved invention, the molten coal activity of extracellular composition, adds Sha Er lake brown coal in the de-cell extract of RM-1 and carries out molten coal experiment.
In 500mL Erlenmeyer flask, add 150mL CZAPECK DOX-medium nutrient solution, cultivate 2 days with 5mL and be inoculated in CZAPECK DOX-medium nutrient solution and continue to cultivate through broken mycelia suspension for every bottle, carry out shake-flask culture with 150r/min shake speed, to guarantee that nutrient solution has sufficient dissolved oxygen to supplement.
Cultivate centrifugation after 7 days, then use organic membrane filtration in 0.5 μ m aperture, the acellular liquid obtaining adds 5g coal sample.After coal sample is dissolved, filtrate darkens, and adopts acid precipitation method to measure transformation efficiency.The results are shown in Table 3:
Table 3: adopt acid precipitation method to measure transformation efficiency result
| Project | Substratum | Substratum+bacterium | Substratum+coal | Substratum+coal+extract |
| Transformation efficiency (%) | 0 | 0 | 1.1 | 54.91 |
Referring to accompanying drawing 3, by above-mentioned experimental verification
davidiella tassianaCGMCCno. 7440 molten coal ability, has obtained efficient, stable technique effect.
Provided by the invention
davidiella tassianacGMCC No. 7440 is a kind of low-rank coal degradation bacteria, by application Sha Er lake brown coal, large South Lake, Hami long-flame coal etc. has same effect, degraded Sha Er lake brown coal have to be stablized and significant effect, cultivate 72 hours its brown coal of can significantly degrading and produce black drop, verify that through many experiments its degradation effect is comparatively stable.
SEQUENCE LISTING
<110> Xinjiang Ke Linside new forms of energy limited liability company
<120> low-rank coal degradation bacteria and the application in brown coal liquefaction thereof
<130> 2013
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 486
<212> DNA
<213> Davidiella tassiana
<400> 1
gttattcata accctttgtt gtccgactct gttgcctccg gggcgaccct gccttcgggc 60
gggggctccg ggtggacact tcaaactctt gcgtaacttt gcagtctgag taaacttaat 120
taataaatta aaacttttaa caacggatct cttggttctg gcatcgatga agaacgcagc 180
gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat catcgaatct ttgaacgcac 240
attgcgcccc ctggtattcc ggggggcatg cctgttcgag cgtcatttca ccactcaagc 300
ctcgcttggt attgggcaac gcggtccgcc gcgtgcctca aatcgtccgg ctgggtcttc 360
tgtcccctaa gcgttgtgga aactattcgc taaagggtgt tcgggaggct acgccgtaaa 420
acaaccccat ttctaaggtt gacctcggat caggtaggga tacccgctga acttaagcat 480
atcaaa 486
Claims (2)
1. a low-rank coal degradation bacteria
davidiella tassianarM-1, low order coal degradation bacteria
davidiella tassianathe preserving number of RM-1 is CGMCC No. 7440.
2. low-rank coal degradation bacteria as claimed in claim 1
davidiella tassianathe application of RM-1 in brown coal liquefaction.
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| 袁红莉,等.不可再生能源物质褐煤的生物可持续发展问题展望-微生物转化与利用研究.《世界科技研究与发展》.2002,(第3期),13-17. |
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